Parental Macronucleus Research Articles

Non-Mendelian, heritable blocks to DNA rearrangement are induced by loading the somatic nucleus of Tetrahymena thermophila with germ line-limited DNA.

Site-specific DNA deletion occurs at thousands of sites within the genome during macronuclear development of Tetrahymena thermophila. These deletion elements are usually not detected in macronuclear chromosomes. We have interfered with the normal deletion of two of these elements, the adjacent M and R elements, by loading vegetative macronuclei with these elements prior to sexual conjugation. Transformed cell lines containing the exogenous M or R element, carried on high-copy-number vectors containing genes encoding rRNA within parental (old) macronuclei, consistently failed to excise chromosomal copies of the M or R element during formation of new macronuclei. Little or no interference with the deletions of adjacent elements or of unlinked elements was observed. The micronucleus (germ line)-limited region of each element was sufficient to inhibit specific DNA deletion. This interference with DNA deletion usually is manifested as a cytoplasmic dominant trait: deletion elements present in the old macronucleus of one partner of a mating pair were sufficient to inhibit deletion occurring in the other partner. Remarkably, the failure to excise these elements became a non-Mendelian, inheritable trait in the next generation and did not require the high copy number of exogenously introduced elements. The introduction of exogenous deletion elements into parental macronuclei provides us with an epigenetic means to establish a heritable pattern of DNA rearrangement.

A Mendelian Mutation Affecting Mating-Type Determination Also Affects Developmental Genomic Rearrangements in Paramecium tetraurelita

In Paramecium tetraurelia, mating type is determined during the differentiation of the somatic macronucleus from a zygotic nucleus genetically competent for both types, O and E. Determination of the developing macronucleus is controlled by the parental macronucleus through an unknown mechanism resulting in the maternal inheritance of mating types. The pleiotropic mutation mtFE affects macronuclear differentiation. Determination for E is constitutive in mutant homozygotes; a number of unrelated mutant characters are also acquired during development. We have examined the possibility that the mutation causes a defect in the developmental rearrangements of the germ-line genome. We show that the excision of an IES (internal eliminated sequence) interrupting the coding sequence of a surface antigen gene is impaired in the mutant, resulting in an alternative macronuclear version of the gene. Once established, the excision defect is indefinitely transmitted across sexual generations in the cytoplasmic lineage, even in a wild-type genetic context. Thus, the processes of mating-type determination and excision of this IES, in addition to their common sensitivity to the mtFE mutation, show a similar maternal inheritance of developmental alternatives in wild-type cells, suggesting a molecular model for mating-type determination.

Effects of nullisomic chromosome deficiencies on conjugation events in Tetrahymena thermophila: insufficiency of the parental macronucleus to direct postzygotic development.

Conjugation fails postzygotically after mating of Tetrahymena cells that have wild-type parental macronuclei but harbor noncomplementing nullisomic parental germline deficiencies. Failures begin shortly after formation of the new macronuclear precursor (anlage) and completion of the first step in elimination of the parental macronucleus (pycnosis). Conjugants fail to complete pair separation, to eliminate one new micronucleus, and to amplify anlage DNA, and they eventually die. Some deficiencies block resorption of the pycnotic parental macronucleus, but we find no evidence for its regeneration. Some deficiencies cause aberrant anlage DNA loss. Those that do not cause DNA loss are epistatic to those that do, indicating that normal anlage development requires the dependent function of at least two types of genes. The possibility that these genes are involved in developmentally regulated anlage DNA rearrangements is discussed. Each observed conjugation defect indicates insufficiency of the parental macronucleus to direct postzygotic development and can be explained by the deficiency of essential conjugation genes that are expressed from the anlage. The failure of nullisomic conjugants to complete pair separation indicates a requirement for gene products, expressed from the early anlage or its precursors, soon after anlage first differentiate.

Identification of a Novel Polypeptide Involved in the Formation of DNA-Containing Vesicles during Macronuclear Development in Tetrahymena

An abundant phosphoprotein with an apparent molecular mass of 65 kDa (p65) has been identified that is enriched in developing new macronuclei (or anlagen) isolated from the holotrichous ciliate, Tetrahymena thermophila. During early stages of macronuclear development, p65 is actively synthesized and deposited into young (4C) anlagen and is not found in micronuclei or parental macronuclei. p65 is not detected in older (8C) anlagen or in vegetatively growing or starved cells, and thus p65 is under stringent developmental control. In situ analyses, using polyclonal antibodies generated against p65, demonstrate that p65 undergoes a pronounced change in distribution during anlagen differentiation. Initially, anlagen are uniformly stained with these antibodies. However, following separation of conjugants, this staining pattern converts to one that is punctate and fragmented. As development proceeds, most, if not all, of these p65-based particles become peripherally located in anlagen and appear as well-defined vesicles surrounding a discrete central core of DNA of yet undetermined origin. This remarkable cytological distribution suggests an involvement of p65 in the elimination or processing of DNA during anlagen differentiation. If the above is correct, p65 provides the first inroad into the protein machinery involved in the well-known DNA rearrangements that characterize ciliate macronuclear development.

Programmed nuclear death: Apoptotic-like degradation of specific nuclei in conjugating Tetrahymena

During conjugation in the ciliated protozoan Tetrahymena, new macronuclei differentiate from germinal zygotic micronuclei while parental (old) macronuclei are eliminated in two stages, condensation or pycnosis coincident with cessation of transcription followed by resorption. We show that pycnosis is accompanied by degradation of old macronuclear DNA into oligonucleosome-sized fragments, a hallmark of programmed cell death, or apoptosis, in a variety of eukaryotic systems. As expected, oligonucleosome formation does not occur in the new micro- and macronuclei, confirming the coordination of different developmental fates for different nuclei in a common cytoplasm. NULLI 3 conjugants have wild-type old macronuclei but lack chromosome 3 germinally and hence in the new macronucleus. In NULLI 3 conjugants, old macronuclear pycnosis and oligonucleosome fragmentation occur normally but the resorption step fails, and the pycnotic old macronucleus is retained, demonstrating that the two steps are genetically separable and thus distinct and implying that genes on chromosome 3 in the new macronucleus are required for the resorption step. Comparison of whole cell polypeptides synthesized during stages of macronuclear development in both wild-type and NULLI 3 crosses reveal similar profiles. However, a polypeptide (apparent M r of 53 kDa) synthesized during old macronuclear elimination is not observed in NULLI 3 conjugants; its role, if any, in elimination of the old macronucleus is unknown. The results show that the old macronucleus is selectively destroyed by a mechanism which is remarkably similar to apoptosis in other eukaryotes and that the zygotic genome is required for the resorption step.

Proteolytic removal of core histone amino termini and dephosphorylation of histone H1 correlate with the formation of condensed chromatin and transcriptional silencing during Tetrahymena macronuclear development.

During the sexual cycle in Tetrahymena, the germ-line micronucleus gives rise to new macro- and micronuclei, whereas the former somatic macronucleus ceases transcription, becomes highly condensed, and is eventually eliminated from the cell. With polyclonal antibodies specific for acetylated forms of histone H4, immunofluorescent analyses have demonstrated that transcriptionally active macronuclei stain positively at all stages of the life cycle except during conjugation, when parental macronuclei become inactive and are eliminated from the cell. In this report using affinity-purified antibodies to either the acetylated or unacetylated amino-terminal domain of H4, immunofluorescent analyses suggest that the acetylated amino-terminal tails of H4 are proteolytically removed in "old" macronuclei during this period. This suggestion was further confirmed by biochemical analysis of purified old macronuclei that revealed several polypeptides with molecular mass 1-2 kD less than that of intact core histones. These species, which are unique to old macronuclei, are not newly synthesized and fail to stain with either acetylated or unacetylated H4 antibodies. Microsequence analysis clearly shows that these polypeptides are proteolytically processed forms of core histones whose amino-terminal "tails" (varying from 13 to 21 residues) have been removed. During the same developmental period, histone H1 is dephosphorylated rapidly and completely in old macronuclei. These results strongly suggest that the developmentally regulated proteolysis of core histones and dephosphorylation of histone H1 participate in a novel pathway leading to the formation of highly condensed chromatin and transcriptional silencing during Tetrahymena macronuclear development.

Antibodies specific to acetylated histones document the existence of deposition- and transcription-related histone acetylation in Tetrahymena.

In this study, we have constructed synthetic peptides which are identical to hyperacetylated amino termini of two Tetrahymena core histones (tetra-acetylated H4 and penta-acetylated hv1) and used them to generate polyclonal antibodies specific for acetylated forms (mono-, di-, tri-, etc.) of these histones. Neither of these antisera recognizes histone that is unacetylated. Immunoblotting analyses demonstrate that both transcription-related and deposition-related acetate groups on H4 are recognized by both antisera. In addition, the antiserum raised against penta-acetylated hv1 also recognizes acetylated forms of this variant. Immunofluorescent analyses with both antisera demonstrate that, as expected, histone acetylation is specific to macronuclei (or new macronuclei) at all stages of the life cycle except when micronuclei undergo periods of rapid replication and chromatin assembly. During this time micronuclear staining is also detected. Our results also suggest that transcription-related acetylation begins selectively in new macronuclei immediately after the second postzygotic division. Acetylated histone is not observed in new micronuclei during stages corresponding to anlagen development and, therefore, histone acetylation can be distributed asymmetrically in development. Equally striking is the rapid turnover of acetylated histone in parental macronuclei during the time of their inactivation and elimination from the cell. Taken together, these data lend strong support to the idea that modulation of histone acetylation plays an important role in gene activation and in chromatin assembly.

Nuclear Roles in the Post‐Conjugant Development of the Ciliate Euplotes aediculatus. III. Roles of Old Macronuclear Fragments in Nuclear and Cortical Development1

Following conjugation of the hypotrichous ciliate Euplotes aediculatus, the posterior fragments of the old (prezygotic) macronucleus persist until after the first vegetative division. These fragments remain viable during exconjugant development as shown by their ability to regenerate should the cell's new macronucleus be damaged. It thus seemed possible that these parental nuclear fragments might participate in the development of the new macronucleus and/or the crucial post‐conjugant cortical reorganization that restores the exconjugant cell's ability to feed. This idea was tested by damaging the posterior fragments with various doses of microbeam ultraviolet (UV) light and assessing the results of such treatment on subsequent cortical and nuclear development. When the posterior fragments of the macronucleus were irradiated at the beginning of cortical morphogenesis, the new macronucleus in 1/3 to 1/2 of the cells assumed a “folded” appearance but did not mature. These cells did not undergo cortical reorganization. Cells irradiated at earlier stages did not detectably develop an oral apparatus; their new macronucleus remained arrested at the spherical anlage stage. The results show that the posterior fragments of the parental macronucleus are necessary for normal nuclear and cortical development. These old nuclear fragments appear to influence the growing macronuclear anlage directly and probably the cortex as well. There also appears to be an information flow from the non‐irradiated partner of a persistently joined exconjugant doublet to its irradiated counterpart, enabling normal anlage and cortex development in the irradiated cell.

Conditional lethality associated with macronuclear development in Tetrahymena thermophila

The development of progeny macronuclei is conditionally lethal in wild-type Tetrahymena thermophila. This lethality is expressed at 40°C and is restricted to those cells producing new macronuclei. The temperature-sensitive period begins at 9 hr and ends at 12.5 hr after conjugation is initiated by the mixing of the prestarved parental strains. This period is coincident with early macronuclear development and the destruction of the parental macronucleus. Our data suggest that an essential, temperature-sensitive step in conjugation is executed at the end of the temperature-sensitive period. Our data also indicate that the cells' commitment to macronuclear development is irrevocable by this point in conjugation.

Downward regulation of macronuclear DNA content in Paramecium tetraurelia: Effects of excess DNA on the subsequent DNA and protein content and the cell growth rate

Paramecium cells were selected which received the entire parental macronucleus at fission and thus started the cell cycle with twice the normal post-fission DNA content. During each of the subsequent two cell cycles the cells synthesized approximately as much DNA as did control cells. The amount of excess macronuclear DNA was consequently halved during each cell cycle. The minimum pre-fission DNA content was just larger than the mean post-replication DNA amount, confirming that a similar amount of DNA, approximately equal to the mean post-fission DNA content of the non-selected population, was synthesized in macronuclei, regardless of the post-fission DNA content. These observations confirm a model for DNA content regulation previously devised for Paramecium and are inconsistent with DNA content regulation schemes proposed for other ciliates. The increased DNA content has no effect either on the subsequent total protein content of pre-fission cells, or on the rate of cell growth. This suggests that the rate of cell growth is limited by the size of the cell when the macronuclear gene-dosage is equal to or greater than that in normal cells. The results also suggest that the amount of DNA synthesized within an interfission period is also limited by the size of the cell and is proportional to the cell mass. Paramecium does not require a fixed nucleocy oplasmic ratio as a pre-condition either for cell division, or, by inference, for initiation of DNA synthesis.